The DII-A/DII-B bi-gene cluster of the model chordate dona intestinalis is one of the most compact and organizationally simple examples of a developmental gene cluster known. While some of the regulatory interactions within gene clusters have been analyzed, the simple DII-A/DII-B cluster in C. intestinalis offers an opportunity to obtain a more complete picture of the coordinated regulatory architecture of a whole developmental gene cluster. The first specific aim of this project is to construct a dual reporter transgene to assay the regulatory output of each of the two clustered gene promoters simultaneously. This transgene will then form the basis for deletion experiments to identify the major genomic regulatory elements acting on either or both promoters. Along with this work will be tests of individual reporter transgenes that can assess the possible regulatory activity of internal portions of the non-coding DMA, such as the intergenic region. A second aim, which will support the first, is the determination of the endogenous transcript expression pattern using whole mount in-situ hybridization. The third specific aim is to examine the specificity and positional importance of selected regulatory elements by replacing or moving them within the overall construct. One set of experiments will replace one of the basal promoters with a copy of the other to test whether each basal promoter responds differently to the genomic environment. Another set of experiments will move regulatory modules within the cluster to test the importance of position on the regulatory output. C. intestinalis was chosen as the model organism because of extensive genomic and developmental resources, and because they are ideally suited to the rapid assessment of gene regulation using transgenic reporters in a small-scale research setting. The long-term objective is to contribute to the database of types and modes of genomic regulation, which will help in understanding the molecular genetic basis of human developmental disorders.